Cardiac resynchronization sensitizes the sarcomere to calcium by reactivating GSK-3β
Jonathan A. Kirk, Ronald J. Holewinski, Viola Kooij, Giulio Agnetti, Richard S. Tunin, Namthip Witayavanitkul, Pieter P. de Tombe, Wei Dong Gao, Jennifer Van Eyk, David A. Kass
Jonathan A. Kirk, Ronald J. Holewinski, Viola Kooij, Giulio Agnetti, Richard S. Tunin, Namthip Witayavanitkul, Pieter P. de Tombe, Wei Dong Gao, Jennifer Van Eyk, David A. Kass
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Research Article Cardiology

Cardiac resynchronization sensitizes the sarcomere to calcium by reactivating GSK-3β

Abstract

Cardiac resynchronization therapy (CRT), the application of biventricular stimulation to correct discoordinate contraction, is the only heart failure treatment that enhances acute and chronic systolic function, increases cardiac work, and reduces mortality. Resting myocyte function also increases after CRT despite only modest improvement in calcium transients, suggesting that CRT may enhance myofilament calcium responsiveness. To test this hypothesis, we examined adult dogs subjected to tachypacing-induced heart failure for 6 weeks, concurrent with ventricular dyssynchrony (HFdys) or CRT. Myofilament force-calcium relationships were measured in skinned trabeculae and/or myocytes. Compared with control, maximal calcium-activated force and calcium sensitivity declined globally in HFdys; however, CRT restored both. Phosphatase PP1 induced calcium desensitization in control and CRT-treated cells, while HFdys cells were unaffected, implying that CRT enhances myofilament phosphorylation. Proteomics revealed phosphorylation sites on Z-disk and M-band proteins, which were predicted to be targets of glycogen synthase kinase-3β (GSK-3β). We found that GSK-3β was deactivated in HFdys and reactivated by CRT. Mass spectrometry of myofilament proteins from HFdys animals incubated with GSK-3β confirmed GSK-3β–dependent phosphorylation at many of the same sites observed with CRT. GSK-3β restored calcium sensitivity in HFdys, but did not affect control or CRT cells. These data indicate that CRT improves calcium responsiveness of myofilaments following HFdys through GSK-3β reactivation, identifying a therapeutic approach to enhancing contractile function.

Authors

Jonathan A. Kirk, Ronald J. Holewinski, Viola Kooij, Giulio Agnetti, Richard S. Tunin, Namthip Witayavanitkul, Pieter P. de Tombe, Wei Dong Gao, Jennifer Van Eyk, David A. Kass

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Figure 2

CRT globally restores myofilament function.

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CRT globally restores myofilament function. (A) Force-calcium data and f...
(A) Force-calcium data and fitted curves for myocytes from the LV lateral wall for controls (black circles, n = 15 myocytes from 6 dogs), HFdys (white triangles, n = 16 myocytes from 6 dogs), and CRT (gray squares, n = 16 myocytes from 7 dogs). Inset: force normalized to Fmax to visualize the change in calcium sensitivity. (B) Fmax is decreased by approximately 50% in HFdys and restored by CRT. HFdys causes desensitization to calcium (increase in EC50), but is restored to control levels with CRT. (C) Force-calcium data and fitted curves for RV trabeculae muscles for control (black circles, n = 14 muscles from 8 dogs), HFdys (white triangles, n = 24 muscles from 13 dogs), and CRT (gray squares, n = 18 muscles from 10 dogs). Inset: force normalized to Fmax to visualize the change in calcium sensitivity. (D) Fmax and EC50 were altered in the RV to an extent similar to that in the LV. (E) Tension cost, the ratio of ATPase activity to developed tension, was reduced in both HF models, and not recovered by CRT. (F) Cross-bridge turnover dynamics (ktr) were also reduced in the HF models and not restored by CRT. (G) Finally, stiffness was not affected in any of the groups. *P < 0.05 vs. control; #P < 0.05 vs. HFdys by 1-way ANOVA.